Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

Free Radical Scavenging and Antioxidative Properties of ‘Silibin’ Complexes on Microsomal Lipid Peroxidation

The antioxidant properties of silibin complexes, the water-soluble form silibin dihemisuccinate (SDH), and the lipid-soluble form, silibin phosphatidylcholine complex known as IdB 1016, were evaluated by studying their abilities to react with the superoxide radical anion (O₂^.−), and the hydroxyl radical (OH^.). In addition, their effect on pulmonary and hepatic microsomal lipid peroxidation had been investigated. Superoxide radicals were generated by the PMS-NADH system and measured by their ability to reduce NBT. IC₅₀ concentrations for the inhibition of the NBT reduction by SDH and IdB 1016 were found to be 25 μM and 316 μM respectively. Both silibin complexes had an inhibitory effect on xanthine oxidase activity. SDH reacted rapidly with OH^. radicals at approximately diffusion controlled rate and the rate constant was found to be (K=8·2×10⁹ M ⁻¹ s⁻¹); it appeared to chelate Fe²⁺ in solution. In hepatic microsomes, when lipid peroxidation was induced by Fe²⁺, SDH inhibited by 39·5 per cent and IdB 1016 by 19·5 per cent, whereas when lipid peroxidation was induced by CuOOH, IdB 1016 exerted a better protective effect than SDH (29·4 per cent and 19·4 per cent inhibition, respectively). In both microsomal systems lipid peroxidation proceeded through a thiol depletion mechanism which could be restored in the presence of silibin complexes. Low levels of lipid peroxidation in pulmonary microsomes point out the differences between in-vitro lipid peroxidation occurring in microsomes of different tissues. The results support the free radical scavenger and antioxidative properties of silibin when it is complexed with a suitable molecule to increase its bioavailabilty. © 1997 John Wiley & Sons, Ltd.     

Non-Enzymic Lipid Peroxidation in Microsomes and Microsomal Phospholipids Induced by Anthracyclines

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes.

Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation.

Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system.

The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.     

Silybin and its bioavailable phospholipid complex (IdB 1016) potentiate in vitro and in vivo the activity of cisplatin

In this study we investigated whether the flavonoid silybin and its bioavailable derivative IdB 1016 (silipide) could enhance the antitumour activity of cisplatin (CDDP), the most commonly used drug in the treatment of gynaecological malignancies. Silybin alone up to 10 (M was unable to produce a relevant in vitro growth inhibition of A2780 cells, whereas CDDP was effective, giving an IC50 value of 0.5+/-0.14 microM. When silybin was combined with CDDP, a dose-dependent and statistically significant (p<0.05) increase of the CDDP activity was noticed, yielding IC50 values of 0.35+/-0.07 and 0.263+/-0.004 microM at silybin concentrations of 1 and 10 microM, respectively. The same trend was observed for in vivo experiments. IdB 1016 alone (1350 mg/kg) did not significantly affect tumour growth, whereas CDDP at the Maximum Tolerated Dose (12 mg/kg) produced a tumour weight inhibition (TWI%) of 80% and a log10 cell kill (LCK) of 0.7. Administration of both drugs resulted in a potentiation of the antitumour activity and TWI% and LCK increased to 90% and 1, respectively. Interestingly, mice receiving the combination recovered earlier in terms of body weight loss as compared to CDDP-treated mice. CDDP at 6 mg/kg yielded TWI of 44% and LCK of 0. The concomitant administration of IdB 1016 (1800 mg/kg) enhanced CDDP anti-tumour activity, with 68% TWI and 0.6 LCK. Finally, an antiangiogenic effect of IdB 1016 in an in vivo experimental model was demonstrated. Median haemoglobin value for the Matrigel from the vehicle-treated controls was 2.43 versus a value of 0.321 for the IdB 1016-treated animals.     

Failure of Electron Paramagnetic Resonance Spectroscopy Studies to Detect Elevated Free Radical Signals in Liver Biopsy Specimens from Patients with Alcoholic Liver Disease

Electron paramagnetic resonance spectroscopy (EPR) was used to study free radicals and transition metal complexes in liver tissue taken from patients with liver disease. Samples were frozen to 77K directly following biopsy to prevent deterioration. Our major aim was to compare signals from patients suffering from alcohol abuse with those from patients having liver damage not induced by alcohol. Samples were obtained from 19 chronic alcohol abusers and 7 non-alcoholic liver disease patients. Of the 19 alcoholic patients, 18 had an increased fat content, 6 had Mallory's hyaline, 12 had an acute inflammatory response, 9 had increased stainable iron and 4 had evidence of fibrosis. A signal derived from free radicals with a spectroscopic splitting factor of g = 2.0045 was found in all samples. This signal in the alcoholic patients had a mean amplitude of 2.96 cm (± 1.42 SD), and in patients with non-alcoholic liver disease 2.12cm (±0.82) (p = 0.10NS), measured under identical instrument settings.

The molar proportion of diene conjugated linoleic acid (DCLA), a free radical marker, in the sera of alcoholic patients was 2.68% (±1.93), but did not correlate with the free radical signals obtained by EPR spectroscopy. Also, there was no correlation between the free radical derived EPR signal and fat content, Mallory's hyaline, inflammatory infiltrate, iron or fibrosis in the liver biopsy specimens. Similarly the concentrations of aspartate transaminase, albumin, and gamma-glutamyl transferase in serum samples showed no correlations with free radical concentrations.

The absence of any significant increase in the stable free radical signal in the presence of alcohol induced liver disease and the lack of correlation between the signal and either histological or serological evidence of liver damage, suggests that alcohol derived free radicals may not be involved in the pathogenesis of alcoholic liver disease.

Unusually large sextet features characteristic of MN(II) complexes were observed for all liver samples. Such signals are very rare in human tissue, showing that there is a strong accumulation of Mn (II) in the liver. However, no systematic trends were observed. In some samples signals characteristic of iron-sulphur cluster units were detected, but again no correlations could be discovered.     

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.

AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.

Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.     

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: an in vitro comparative study

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.     

Free Radicals Appear to Affect Survival of Hepatocytes at 4°C

1) Rat hepatocytes, stored in a simple salts medium for 24 h at 4°C, retain more than 80% of their capacity to synthesize glucose from lactate.

2) The combination of NH₄Cl with oleate is cytotoxic during storage and during subsequent incubation of hepatocytes from 48 h starved rats, but not to hepatocytes from fed rats.

3) Protection against cytotoxicity is afforded by albumin and by a number of other compounds, notably polyols and glycerol.

4) These compounds appear to exert their effects by scavenging free radicals and, in the case of polyols and glycerol, by supplying reducing equivalents to maintain the redox state of the cell in the face of increased flux through glutathione peroxidase.     

Spin-trapping of free radicals formed during in vitro and in vivo metabolism of 3-methylindole

Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.     

Free Radicals in the Atmosphere

Like the oxidation in a flame, the oxidation in the atmosphere is mediated by free radicals. Unlike a flame, however, atmospheric oxidation needs an external source of energy: the sun light. In fact the most important radical acting in the lower atmosphere, the hydroxyl radical, OH, is produced following the UV-photolysis of ozone, O,which yields an excited oxygen atom, O'D:

OH reacts with most atmospheric trace gases, in many cases as the first and rate determining step in the reaction chain leading to oxidation. In this way a host of various other radicals (e.g. peroxy radicals), most of them very short lived, are generated. Usually these oxidation reactions form chains which regenerate OH, thus maintaining OH at a relatively high concentration level on the order of 10⁶cm∼³ during the day. The reactions which control the OH concentration will be discussed in detail. During the night radical formation is greatly diminished. It proceeds, for example, through the reaction of defines with O, and. in dry air, through reaction of defines and aldehydes with the nitrate radical, NO,.     

Spin Trapping of Inorganic Radicals

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals.

Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (·N_j· ·CN, ·SCN) are discussed in more detail.     

The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

Copper-Ligand Interactions and Physiological Free Radical Processes. pH-Dependent Influence of Cu2+ Ions on Fe2+-Driven OH- Generation

Prior to comparative studies on the reactivity of various copper complexes with respect to OH radicals, the influence of free Cu²⁺ ions on the superoxide-independent generation of OH radicals through Fenton assays and water gamma radiolysis has been tested in the present work.

Cu²⁺ ions have been shown to behave in a distinct manner towards each of these two production systems. As was logically expected from the noninvolvement of copper in OH^- radical production through gamma radioiysis, no influence of Cu²⁺ ions has been observed on the amount of radicals detected in that case. In contrast, Cu²⁺ ions do influence OH^- radical generation through iron-driven Fenton reactions, but differently depending on copper concentration.

When present in high concentrations, Cu²⁺ ions significantly contribute to OH^- radical production, which confirms previous observations on the reactivity of these in the presence of hydrogen peroxide. At lower levels corresponding to copper/iron ratios below unity on the contrary, Cu²⁺ ions behave as inhibitors of the OH^- production in a pH-dependent manner over the 1-6 range investigated: the lower the pH, the greater the inhibition.

The possible origin of this previously unreported inhibitory effect is discussed.     

Effect of Daunorubicin on Subcellular Pools of Glutathione in Cultured Heart Cells from Neonatal Rats

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase.

Control heart cells were found to contain 12.2 ± 1.8 nmolesGSH/IO⁶ cells. Daunorubicin causedarapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH.

By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 89 ± 1.5nmoles, 10 cells and 3.3 ± 0.6 nmoles/10⁶ cells. respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and. after a short lag period. a release of lactate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH.

No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase. whereas dithiothreitol offered partial protection.

The results provide support for an oxidative mechanism in which the decrease in the cytosolic pool of GSH may be the causative factor of daunorubicin-induced toxicity. This decrease in GSH may affect the cytosolic NADPH and various redox groups on proteins, thereby altering the permeability of the plasma membrane and finally causing cell damage.     

Plasma Lipid Peroxides in Murine Sepsis —Sex Differences and Effect of Antioxidative/Anti-Inflammatory Therapy

In order to investigate the influence of antioxidative anti-inflammatory combination therapy (AACT) with dimethyl sulfoxide (DMSO). chlorpromaittic (CPZ) and vitamin E upon the activity of the inflammation. plasma lipid peroxide was measured as thiobarbituric acid reactive substance (TBARS) 12hrs postoperatively in the moclitied cecal ligation sepsis model in the mouse.

Significantly higher TBARS levels were found in the male control group (13.7 ± 0.7nmol MDA/ml) than in the female control group (11.6 ± 0.6nmol MDA/ml).

The operated male group had significantly higher TBARS levels (16.2 ± 0.6 nmol MDA/ml) than the unoperdted male control group (13.7 ± 0.7nmol MDA/ml). No increase of TBARS levels was observed in the operated female group.

Both male and female operated group. when postoperatively treated with AACT had the same TBARS level as the not operated male or female control group.

Survival curves of operated male and female group did not demonstrate any significant difference. The survival was better in an operated male and an operated female group. when postoperatively treated with AACT.

It was concluded that the applied TBARS test IS too insensitive to follow the activity of the inflammation and has no predictive value for the outcome of sepsis in this model.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Ciprofloxacin-induces free radical production in rat cerebral microsomes

In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone by electron spin resonance spectroscopy. Active microsomes, dihydronicotinamide-adenine dinucleotide phosphate, and ciprofloxacin were necessary for the formation of a spin trap/radical adduct. Adduct formation increased dose-dependently at 0.5–1?mM CPFX concentration for 180?min, and 0.3–1 mM concentration level for 240?min. The addition of SKF 525A, ZnCl₂ or desferrioxamine to the incubation system caused complete inhibition of the radical formation. However, pretreatment of microsomal system with superoxide dismutase (SOD) did not induce any protective effect. Induction of lipid peroxidation, and depletion of thiol levels by CPFX were also shown in the system. These results strongly suggested that CPFX produces free radical(s) in the cerebral microsomes of rats.     

Cell-Dependent Chemiluminescence. Modulation of the N-Formyl Chemotactic Peptide (Fnlpntl) Mediated Oxidative Burst in Human Polymorphonuclear Leukocytes (Pmnl) By Murine Monoclonal Antibody Nms-1

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL.

We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL ± moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition.

Stimulation of human PMNLs by 10nM FNLPNTL induced a rapid burst of chemiluminescence which peaked ∼5min after stimulus addition. The subsequent addition of moAB NMS-1 (≥2μg/ml DPBS(+)—0.1% HSA, 37°C) to FNLPNTL-stimulated PMNLs—after the FNLPNTL-mediated response had already decayed (16-18 min) - without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent.

Treatment of PMNLs with moAB NMS-1 (≥ 1μg/ml DPBS(+)—0.1% HSA, 3 min, 37°C)—prior to FNLPNTL (10nM) stimulation - increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak ∼16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the respone was not changed compared to controls which received no moAB NMS-1 treatment.

The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (5-60 min), persistent activation was not detected.     

Comparison of the Inactivation of Microsomal Glucose-6-Phosphatase by in Situ Lipid Peroxidation-Derived 4-Hydroxynonenal and Exogenous 4-Hydroxynonenal

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied.

2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes.

3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E.

4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5μM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150μM or 300μM was needed with a preincubation time of 30 and 60 min, respectively.

5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynbnenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.     

Free radical scavenging and copper chelation: a potentially beneficial action of captopril

Captopril (CpSH), an angiotensin converting enzyme (ACE) inhibitor, is reported to provide protection against free-radical mediated damage. The purpose of this study was to investigate, by means of pulse radiolysis technique, the behaviour of CpSH towards radiation-induced radicals in the absence and in the presence of copper(II) ions, which can play a relevant role in the metal catalysed generation of reactive oxygen species. The results indicate that the -SH group is crucial in determining the radical scavenging action of CpSH and the nature of the resulting CpSH transient products in the absence or in the presence of oxygen.

In the presence of Cu(II), the -SH group is still involved in the biological action of the molecule participating both in the one-electron reduction of Cu(II) with formation of CpSSCp, and in Cu(I) chelation. This conclusion is supported by the Raman spectroscopic data which allow to identify the CpSH sites involved in the copper complex at different pH.

These results suggest that CpSH may potentially inhibit oxidative damage both through free radical scavenging and metal chelation. Considering the low CpSH concentration in vivo, the metal chelation mechanism, more than the direct radical scavenging, could play the major role in moderating the toxicological effects of free radicals.